Antiplasmodial Compounds from Deep-Water Marine Invertebrates

Novel drug leads for malaria therapy are urgently needed because of the widespread emergence of resistance to all available drugs. Screening of the Harbor Branch enriched fraction library against the Plasmodium falciparum chloroquine-resistant strain (Dd2) followed by bioassay-guided fractionation led to the identification of two potent antiplasmodials; a novel diterpene designated as bebrycin A (1) and the known C21 degraded terpene nitenin (2). A SYBR Green I assay was used to establish a Dd2 EC50 of 1.08 ± 0.21 and 0.29 ± 0.02 µM for bebrycin A and nitenin, respectively. Further analysis was then performed to assess the stage specificity of the inhibitors antiplasmodial effects on the Dd2 intraerythrocytic life cycle. Exposure to bebrycin A was found to block parasite maturation at the schizont stage if added any time prior to late schizogony at 42 hours post invasion, (HPI). In contrast, early life cycle exposure to nitenin (prior to 18 HPI) was identified as crucial to parasite inhibition, suggesting nitenin may target the maturation of the parasite during the transition from ring to early trophozoite (6–18 HPI), a novel property among known antimalarials.


Introduction
Worldwide, malaria continues to be a prevalent infectious disease with an estimated 209 million clinical cases in 2019, with children and pregnant women being most at risk [1,2]. There remain limited treatment options due to the widespread prevalence of drug resistance among the disease causing Plasmodium spp. parasite. Even artemisinin-based combination therapies (ACTs), which are the front-line therapeutic options for uncomplicated Plasmodium falciparum malaria, are showing signs of ineffectiveness in a wide area of Southeast Asia due to point mutations in kelch13 [3][4][5]. In addition, parasites are exhibiting signs of resistance against artemisinin partner drugs [6,7]. This grim situation underscores the urgent need to develop novel antimalarials acting on targets different from existing therapeutics. Marine organisms have long been a source of novel natural products with unique chemical scaffolds possessing a variety of potent biological activities [8]. This rich marine biodiversity provides us an enormous opportunity to identify novel antimalarial leads from specialized metabolites of marine organisms [9].
The Harbor Branch Oceanographic Institute of Florida Atlantic University has a substantial collection of marine invertebrates, many collected in deep-water habitats using manned submersibles. A sub-set of chemically rich organisms in the collection has been fractionated using medium pressure liquid chromatography to create a library of enriched fractions [10]. Screening of this library for antiplasmodial activity led to the identification been fractionated using medium pressure liquid chromatography to create a library of enriched fractions [10]. Screening of this library for antiplasmodial activity led to the identification of 165 fractions from 85 taxonomically distinct organisms that inhibit the proliferation of the parasite at concentrations of ≤5 µg/mL [11]. This manuscript describes the isolation, structure elucidation, and biological activity of two classes of compounds identified from this screening effort. They include a novel diterpene which we have designated as bebrycin A (1) from the octocoral Bebryce grandis [12], and the previously reported C-21 degraded terpenoid nitenin (2) [13,14] from a specimen of Spongia lamella ( Figure 1). Fractions from both organisms showed substantial activity against the Dd2 chloroquine resistant strain of P. falciparum and were selected for further fractionation and structure elucidation.

Chemical Analysis
The sample of Bebryce grandis [Phylum: Cnidaria, Class: Anthozoa, Subclass: Octocorallia, Order: Alcyonacea, Family: Plexauridae] from which bebrycin A (1) was isolated was collected using the Johnson-Sea-Link II submersible at a depth of 131 m off Ocean Cay, Bahamas, approximately 20 nautical miles south of Bimini. The sample was frozen at −20 °C immediately after collection and stored frozen until work-up. The frozen octocoral was extracted exhaustively with ethanol (EtOH). After concentration by distillation under reduced pressure, the extract was partitioned between n-butanol and H2O. The n-butanol partition was fractionated by medium pressure liquid chromatography (MPLC) using a Combi-Flash Rf4x and a Redi-Sep Gold C-18 column eluted with a linear gradient of CH3CN and H2O. Final purification was achieved using semi-preparative HPLC on a Vydac C-18 column with isocratic elution (CH3CN:H2O (55:45 v/v)) to yield 1 (1.9 mg, 1.75 × 10 −3 % of wet weight) as an amorphous white solid. Two additional specimens were also found to contain 1 and were used to isolate additional material for bioassays.
Inspection of the 13 C NMR spectrum coupled with high resolution electrospray ionization mass spectrometry (HR-ESIMS) analysis of 1 suggested a molecular formula of C20H32O2 requiring 5 degrees of unsaturation ( Figures S5 and S23). A strong absorption observed at 1705 cm −1 in the IR spectrum suggested the presence of conjugated ketone functionality in the molecule. The 13 C NMR spectra observed in d4-methanol showed the presence of two resonances attributable to ketones (δC 211.8 and 206.7); three olefinic methine carbons (δC 157.3, 126.9 and 126.5); and one quaternary olefinic carbon (δC 132.8).
No additional unsaturation was apparent from the NMR spectra and therefore the structure of 1 was assigned one ring. The 1 H NMR and edited g-HSQC ( Figures S4, S6 and S7) confirmed the presence of the two double bonds, as well as identified two sp 3 hybridized methine carbons, six methylene groups, and five methyl groups in 1. Interpretation of the 2D g-DQF COSY spectrum (Figures S8-S10) allowed for the assignment of three spin systems in 1 (bolded bonds in Figure 2) along with an isolated methylene group appearing as an AX pattern (δH 3.00 d (J = 12.4 Hz) and 2.92 d (J = 12.4 Hz)). Data

Chemical Analysis
The sample of Bebryce grandis [Phylum: Cnidaria, Class: Anthozoa, Subclass: Octocorallia, Order: Alcyonacea, Family: Plexauridae] from which bebrycin A (1) was isolated was collected using the Johnson-Sea-Link II submersible at a depth of 131 m off Ocean Cay, Bahamas, approximately 20 nautical miles south of Bimini. The sample was frozen at −20 • C immediately after collection and stored frozen until work-up. The frozen octocoral was extracted exhaustively with ethanol (EtOH). After concentration by distillation under reduced pressure, the extract was partitioned between n-butanol and H 2 O. The n-butanol partition was fractionated by medium pressure liquid chromatography (MPLC) using a Combi-Flash R f 4x and a Redi-Sep Gold C-18 column eluted with a linear gradient of CH 3 CN and H 2 O. Final purification was achieved using semi-preparative HPLC on a Vydac C-18 column with isocratic elution (CH 3 CN:H 2 O (55:45 v/v)) to yield 1 (1.9 mg, 1.75 × 10 −3 % of wet weight) as an amorphous white solid. Two additional specimens were also found to contain 1 and were used to isolate additional material for bioassays.
Inspection of the 13 C NMR spectrum coupled with high resolution electrospray ionization mass spectrometry (HR-ESIMS) analysis of 1 suggested a molecular formula of C 20 H 32 O 2 requiring 5 degrees of unsaturation ( Figures S5 and S23). A strong absorption observed at 1705 cm −1 in the IR spectrum suggested the presence of conjugated ketone functionality in the molecule. The 13 C NMR spectra observed in d 4 -methanol showed the presence of two resonances attributable to ketones (δ C 211.8 and 206.7); three olefinic methine carbons (δ C 157.3, 126.9 and 126.5); and one quaternary olefinic carbon (δ C 132.8).
No additional unsaturation was apparent from the NMR spectra and therefore the structure of 1 was assigned one ring. The 1 H NMR and edited g-HSQC ( Figures S4, S6 and S7) confirmed the presence of the two double bonds, as well as identified two sp 3 hybridized methine carbons, six methylene groups, and five methyl groups in 1. Interpretation of the 2D g-DQF COSY spectrum (Figures S8-S10) allowed for the assignment of three spin systems in 1 (bolded bonds in Figure 2) along with an isolated methylene group appearing as an AX pattern (δ H 3.00 d (J = 12.4 Hz) and 2.92 d (J = 12.4 Hz)). Data from the 2D-gHMBC experiment (Figures S11-S15) allowed the spin systems to be tied together as follows (key correlations in the HMBC spectrum are shown in Figure 2). The singlet methyl groups observed at δ H 1.12 and 1.08 (H 3 -16 and H 3 -17) both have strong correlations to the quaternary carbon observed at δ C 39.1 (C-1), the methylene carbon observed at δ C 41.7 (C-15) and the olefinic carbon observed at δ C 157.3 (C-2). They also showed strong correlations to each other δ H 1.12 (H 3 -16) to δ C 26.6 (C-17) and δ H 1.08 (H 3 -17) to δ C 27.4 (C-16), suggesting the presence of geminal methyl groups attached to a quaternary carbon that connects the C-2/C-3 olefin spin system with the C-15 to C-20 spin system. Correlations observed in the 2D-gHMBC spectrum between H-2 and C-1, C-15 and C-16, as well as correlations from H-15ab to C-1, C-2, C-16, and C-17, further support this assignment. The observed chemical shifts for H-2 (δ H 6.83) and H-3 (δ H 6.10), along with long range couplings observed in the g-HMBC spectrum for both H-2 and H-3 to a ketone resonance observed at δ C 206.7 (C-4), allow for incorporation of the first ketone moiety as C-4. This was further extended to incorporate the C-5 to C-10 spin system based upon 1 H-13 C long range couplings observed in the g-HMBC spectrum from H 3 -18 (δ H 1.01) to the C-4 ketone resonance. Correlations from H-10ab (δ H 2.59 and 2.05) to the second ketone carbon observed at δ C 211.8 allowed for placement of the final ketone as C-11. Both protons of an isolated methylene group observed at δ H 3.00 and 2.92 showed strong correlations in the HMBC spectrum to both the C-11 ketone carbon and to the C-10 methylene group, allowing for its assignment as C-12. These protons also showed correlations in the HMBC spectrum to the olefinic carbon C-13 (δ C 132.8), as well as the olefinic methyl C-20 (δ C 17.3), allowing for the final connection and closing of the macrocyclic ring to form a 15 carbon macrocyclic structure. from the 2D-gHMBC experiment (Figures S11-S15) allowed the spin systems to be tied together as follows (key correlations in the HMBC spectrum are shown in Figure 2). The singlet methyl groups observed at δH 1.12 and 1.08 (H3-16 and H3-17) both have strong correlations to the quaternary carbon observed at δC 39.1 (C-1), the methylene carbon observed at δC 41.7 (C-15) and the olefinic carbon observed at δC 157.3 (C-2). They also showed strong correlations to each other [δH 1.12 (H3-16) to δC 26.6 (C-17) and δH 1.08 (H3-17) to δC 27.4 (C-16), suggesting the presence of geminal methyl groups attached to a quaternary carbon that connects the C-2/C-3 olefin spin system with the C-15 to C-20 spin system. Correlations observed in the 2D-gHMBC spectrum between H-2 and C-1, C-15 and C-16, as well as correlations from H-15ab to C-1, C-2, C-16, and C-17, further support this assignment. The observed chemical shifts for H-2 (δH 6.83) and H-3 (δH 6.10), along with long range couplings observed in the g-HMBC spectrum for both H-2 and H-3 to a ketone resonance observed at δC 206.7 (C-4), allow for incorporation of the first ketone moiety as C-4. This was further extended to incorporate the C-5 to C-10 spin system based upon 1 H-13 C long range couplings observed in the g-HMBC spectrum from H3-18 (δH 1.01) to the C-4 ketone resonance. Correlations from H-10ab (δH 2.59 and 2.05) to the second ketone carbon observed at δC 211.8 allowed for placement of the final ketone as C-11. Both protons of an isolated methylene group observed at δH 3.00 and 2.92 showed strong correlations in the HMBC spectrum to both the C-11 ketone carbon and to the C-10 methylene group, allowing for its assignment as C-12. These protons also showed correlations in the HMBC spectrum to the olefinic carbon C-13 (δC 132.8), as well as the olefinic methyl C-20 (δC 17.3), allowing for the final connection and closing of the macrocyclic ring to form a 15 carbon macrocyclic structure. The geometry of the C-2-C-3 double bond was assigned to be E-configuration based upon the large coupling constant between H-2 and H-3 (J = 15.8 Hz). The C-13-C-14 double bond was assigned as E configuration due to the observation of an nOe between H3-20 and H-15ab, and between H-14 and H-12ab. Additional support for the E configuration is the chemical shift of C-20 (δC 17.3). Methyl groups on E-configured trisubstituted olefins are observed below 20 ppm [15,16]. Assignment of the relative configuration of the molecule is complicated by the flexibility of the 15-membered ring. C-5 is tentatively assigned as S* based upon the following data: strong nOes are observed between H-5 and both H-2 and H-3 in both the 2D-NOESY and 1D-dpfgse nOe spectra ( Figure 2  The geometry of the C-2-C-3 double bond was assigned to be E-configuration based upon the large coupling constant between H-2 and H-3 (J = 15.8 Hz). The C-13-C-14 double bond was assigned as E configuration due to the observation of an nOe between H 3 -20 and H-15ab, and between H-14 and H-12ab. Additional support for the E configuration is the chemical shift of C-20 (δ C 17.3). Methyl groups on E-configured trisubstituted olefins are observed below 20 ppm [15,16]. Assignment of the relative configuration of the molecule is complicated by the flexibility of the 15-membered ring. C-5 is tentatively assigned as S* based upon the following data: strong nOes are observed between H-5 and both H-2 and H-3 in both the 2D-NOESY and 1D-dpfgse nOe spectra (Figure 2 and Figures S16-S22), suggesting that this proton faces towards the center of the macrocycle. H-2 shows a strong nOe to the olefinic proton H-14 and to the H 2-15 methylene protons, suggesting that these atoms also face towards the center of the macrocyclic ring. The configuration at C-9 has been tentatively assigned as R*, but due to conformational flexibility of the 15 membered ring the data to support this is limited. H-9 appears as an 8 line multiplet consistent with coupling to seven protons, all with similar J couplings of 6 to 7 Hz. In the 2D-NOESY spectrum H 3 -19 has correlations to H-8a, H-10a and H-10b. These nOes suggest that H 3 -19 is in a pseudo-equatorial position. The 15 ring macrocycle has substantial conformational flexibility and the assignments are considered tentative. The absolute configuration has not been assigned. Nitenin (2) was isolated from a sample of Spongia lamella [Phylum: Porifera, Class: Demospongiae, Order: Dictyoceratida, Family: Spongiidae] collected using the Johnson-Sea-Link I submersible, from a rock outcrop on a sand flat, 98.8 m deep, off the east coast of Fuerte Ventura, Canary Islands. The sample was frozen at −20 • C immediately after collection and stored frozen until work-up. The frozen sponge was extracted exhaustively with ethanol:ethyl acetate (EtOH:EtOAc, 1:9 v/v) followed by EtOH. After concentration by distillation under reduced pressure, the combined extracts were partitioned between EtOAc and H 2 O. The EtOAc partition was fractionated by MPLC on a Combi-Flash R f 4x using a Redi-Sep Gold C-18 column and a linear gradient of CH 3 CN and H 2 O. Final purification was achieved using preparative HPLC on a Vydac C-18 column, eluted with a linear gradient of CH 3 CN:H 2 O to yield 2 (2.6 mg, 1.1 × 10 −3 % of wet weight) as a colorless oil. The structure was defined through interpretation of high resolution mass spectrometry data coupled with a full 2D NMR data set and confirmed by comparison to the published data (Supporting Figures S26-S34) [14].

Biological Activity
Bebrycin A and nitenin were assayed for their EC 50 values against the P. falciparum chloroquine-resistant strain (Dd2) using a SYBR Green I fluorescence assay. The EC 50 s were determined to be 1.08 ± 0.21 and 0.29 ± 0.02 µM for bebrycin A and nitenin, respectively. To determine the selectivity of these inhibitors for the malaria parasite, cytotoxicity against the HepG2 human hepatocyte carcinoma cell line was evaluated using a formazan based MTS assay. Bebrycin A gave an EC 50 value against HepG2 of 21.8 ± 1.4 µM for a selectivity index (SI) of 20.1, while nitenin gave an EC 50 of 18.3 ± 1.1 µM, SI = 62.5.
To better define the mechanism of action, the developmental stage specific effects of the compounds during intraerythrocytic maturation of P. falciparum were assessed. Synchronized parasites at the early ring (6 HPI), late ring (18 HPI), and late trophozoite (30 HPI) stages were exposed to a 5 × EC 50 concentration of compound. The microscopical Mar. Drugs 2021, 19, 179 5 of 11 evaluation of the development stage progression (inset) in addition to the flow cytometric analysis of the DNA content with YOYO-1 was then performed. As is evident in Figure 3, maturation of the nitenin treated culture was inhibited if compound was added early, before the transition of ring to early trophozoite stage (6 HPI). In contrast, exposure of the culture to nitenin at 18 HPI did not impair maturation, and the parasites progressed to the ring stage in the next developmental cycle (54 HPI) similar to the control. Similar results were obtained when parasites were treated at 30 HPI ( Figure S35). The early developmental stage specific action of nitenin is significant, as a recent report [17] suggests that only artemisinin and artesunate among antimalarial drugs in clinical use act during the ring stage.
Exposure of the culture to bebrycin A blocked maturation when added at 6, 18, or 30 HPI (Figure 3). Morphologically, parasites treated at 6 HPI did not proceed beyond the late trophozoite-early schizont stages, with no apparent shift in YOYO-1 fluorescence indicative of multinucleation. Treatment at 18 HPI resulted in a schizont like phenotype both morphologically and via the flow cytometric profile. Treatment at 30 HPI also appeared to inhibit during schizogony. However, in contrast to dihydroartemisinin (DHA), which completely inhibits parasite maturation, bebrycin A exposed parasites, although blocked at the schizont stage, appeared to increase in DNA content when treated at 30 HPI. Again, among the current antimalarials, only artemisinin exhibits potent activity at the schizont stage [17]. Therefore, the discovery of nitenin and bebrycin A from marine macroorganisms is significant as these chemotypes act on parasite life cycle stages that are not currently targeted by approved antimalarials other than artemisinin [17]. Exposure of the culture to bebrycin A blocked maturation when added at 6, 18, or 30 HPI (Figure 3). Morphologically, parasites treated at 6 HPI did not proceed beyond the late trophozoite-early schizont stages, with no apparent shift in YOYO-1 fluorescence indicative of multinucleation. Treatment at 18 HPI resulted in a schizont like phenotype both morphologically and via the flow cytometric profile. Treatment at 30 HPI also ap- Figure 3. Nitenin and bebrycin A exhibit distinct profiles of inhibition during P. falciparum intraerythrocytic maturation. Synchronized Dd2 culture was exposed to the test compounds at 5 × EC 50 starting at 6, 18, and 30 HPI and monitored into the next life cycle stage up to 54 HPI. Untreated wells (containing DMSO vehicle) or dihydroartemisinin (DHA) were include as controls. Giemsa smears (inset) and flow cytometry with nucleic acid staining fluorophore YOYO-1 were collected every

Discussion
Previous studies of octocorals belonging to the genus Bebryce have led to the isolation of a number of different classes of organic compounds including carotenoids [18], a sterol glycoside [19], and a guaiazulene [20]. Bebrycin A is a diterpene with a rare C-15 membered carbocyclic ring. It can be envisioned as forming from ring opening of the cyclopropyl group of a casbene class terpenoid, or from rearrangement of a cembranoid to yield the 15 membered ring. Recently, a terpene synthase from a marine Micromonospora has been identified that produces the diterpene micromonocyclol that has a C-15 membered carbocyclic ring as the direct product of its terpene synthase [21]. Genome mining indicated that this terpene cyclase is common amongst marine Micromonospora strains and could potentially be used as a taxonomic marker. Bebrycin A and micromonocyclol are related molecules with different oxidation patterns, and the discovery of this terpene cyclase in marine bacteria opens the possibility that bebrycin A is bacterially produced. Nitenin is a C-21 terpenoid with two beta-substituted furans at distal ends of the molecule. It has been proposed that it is formed through degradation of a larger sesterterpene [13]. Prior reports describing the isolation of nitenin have not reported significant biological activity for nitenin [13,14,[22][23][24]. In the current study, we find that both compounds have potent activity against the chloroquine resistant P. falciparum strain (Dd2), with good selectivity for the parasite over mammalian cell line HepG2. The specific action of nitenin and bebrycin A on the early or late developmental stages respectively is significant as a recent report [17] suggests that only artemisinin and artesunate among antimalarial drugs in clinical use act on the ring stage and only artemisinin exhibits activity on schizonts [17]. Both bebrycin A and nitenin act on parasite life cycle stages that are not currently targeted by antimalarials other than artemisinin. This may open new avenues for the development of novel classes of antimalarial agents as partner drugs for artemisinin combination therapy.

Chemical Analysis
Optical rotation was measured on a Rudolph Research Analytical AUTOPOL III automatic polarimeter. UV spectra were collected on a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). NMR data was collected on a JEOL ECA-600 spectrometer (JEOL USA, Peabody, MA, USA) operating at 600 MHz for 1 H, and 150.9 for 13 C. The edited gHSQC spectrum was optimized for 140 Hz and the gHMBC spectrum optimized for 8 Hz. Chemical shifts were referenced to solvent, e.g., CD 3 OD, δ H observed at 3.31 ppm and δ C observed at 49.1 ppm. High-resolution mass spectrometry for 1 was performed on a JEOL AccuTOF-DART 4G (JEOL USA, Peabody, MA, USA) using the ESI source for ionization and detected in positive ion mode. High-resolution mass spectrometry for 2 was performed on a Thermo Fisher Orbitrap (Thermo Fisher Scientific, San Jose, CA, USA) using the ESI source for ionization and detected in positive ion mode. IR data was collected on a Perkin Elmer Spectrum 100 with Universal ATR (Perkin Elmer, Waltham, MA, USA).

Biological Material
Bebrycin A (1) was isolated from three separate specimens of the octocoral Bebryce grandis (Figures S1 and S2). The primary specimen used in this study was HBOI Sample All three specimens have external morphology and spicules characteristic of the species Bebryce grandis Deichmann, 1936; [Phylum: Cnidaria, Class: Anthozoa, Subclass: Octocorallia, Order: Alcyonacea, Family: Plexauridae]. The species is described in Deichmann (1936) [25], page 125-126, and images are in Bayer and Cairns [26]. The specimens are tan colored, planar with upward curved, stout branches (2 mm diameter), and rounded calyces (1 mm diameter, 1 mm tall), which tend to alternate on the sides. The surface is fine grained, with epifauna including small hollow tubes and Serpulidae worm tubes. The axis is brown and fibrous. The spicules are dominated by cup shaped rosette bodies (0.08 to 0.12 mm tall), and tri-radiate and quad-radiate crosses are about 0.15 to 0.3 mm. It is known from depths of 91 to 281 m, and distribution includes the southeastern U.S., the Gulf of Mexico and the Caribbean.
Nitenin (2) Figure S24). The morphology is lamellate to fan-shaped, with multiple lamellae arising from a common base. Individual lamellae are up to 15 cm wide, 20 cm high, and 5-10 mm thick. The surface of the sponge is microconulose; abundant oscula (1-2 mm wide, 5-10 mm apart) occur on one side of each lamella and on the other side are only ostia. The sponge was yellow/tan alive. The consistency is dense and compressible. The skeleton is fibroreticulate, dominated by secondary clear fibers (20-30 µm in diameter) and less abundant primary fibers (50-100 µm in diameter) that are cored and covered by sand. Secondary fibers form meshes (100-300 µm in diameter). The specimen has been identified as Spongia lamella [Phylum: Porifera, Class Demospongiae Order: Dictyoceratida, Family Spongiidae, Genus/Species Spongia lamella (Schulze, 1879) [27] previously known only from the Mediterranean Sea, the Atlantic coast of Portugal and the Straits of Gibraltar [28]. A taxonomic reference sample is archived at the HBOM, catalog number 003:00839.

Extraction and Isolation
Isolation of bebrycin A (1): Three different samples were shown to have compound 1. All samples were frozen at −20 • C immediately after collection and stored frozen until work-up. An example of the isolation process for organism 10-V-00-1-004 follows. The frozen octocoral (133 g) was extracted exhaustively with ethanol (EtOH) followed by ethanol:ethyl acetate ((EtOH: EtOAc)1:9 v/v). After concentration by distillation under reduced pressure, the extract (2.9 g) was partitioned between n-butanol and H 2 O to yield 0.598 g of butanol partition and 1.7 g of aqueous partition. The n-butanol partition was fractionated by medium pressure liquid chromatography (MPLC) on an Isco Combi-Flash R f 4x (Teledyne ISCO, Lincoln, NE, USA) using a Redi-Sep C 18 column and a linear gradient of CH 3 CN and H 2 O over 20 min. A total of four fractions were subsequently collected. Assay of fraction 3 (34.6 mg) showed activity against P. falciparum and 28.2 mg was further fractionated by semi-preparative HPLC on a Vydac C 18 column (10 × 250 mm, 10 µm particle size) using isocratic elution with CH 3 CN:H 2 O (55:45 v/v) to yield 1 (1.9 mg, 1.7 × 10 −3 % of wet weight) eluting at 19 min.
Isolation of nitenin (2): A 242 g sample of the frozen sponge, 5-VI-91-2-007, was x extracted exhaustively by macerating with EtOH:EtOAc, (1:9, 2 × 250 mL) followed by EtOH (4 × 200 mL) using a Waring blender and filtered through Celite ® (Millipore Sigma, St. Louis, MO, USA). The combined extract was concentrated by distillation under reduced pressure to yield 10.34 g of crude extract. The extract was partitioned between EtOAc and H 2 O (3 × 100 mL portions). The EtOAc partition was concentrated under reduced pressure to yield 1.5 g of a yellow-brown solid. The EtOAc partition was pre-adsorbed onto a small amount of C 18 stationary phase and then separated by MPLC on an Isco Combi-Flash R f 4x on an Isco RfGold ® reverse-phase C 18 column (26 g) using a linear gradient. The   Figure S34.
SYBR Green I Fluorescence Assay: Parasite viability was determined using a SYBR Green I-based fluorescent assay [31][32][33]. Extracts or pure compounds in DMSO were diluted in culture medium and screened at varying concentrations at a maximum DMSO concentration of 0.125%. The diluted fractions/compounds were added to asynchronous Dd2 culture at a 1% parasitemia and 2% hematocrit in black, flat-bottom 96-well plates (Santa Cruz Biotechnology, Dallas, TX, USA). Chloroquine (10 µM) was used as a positive control to determine the baseline fluorescence value. Following a 72 h incubation at 37 • C, the plates were frozen at −80 • C. One hundred microliters of the lysis buffer (20 mM Tris-HCl, 0.08% saponin, 5 mM EDTA, 0.8% Triton X-100, and 0.01% SYBR Green I) was added to each well of the thawed plates. After incubation in the dark at 37 • C for 30 min, the fluorescence emission was measured using a Synergy H4 hybrid multimode plate reader (Biotek, Winooski, VT, USA) set at 485 nm excitation and 530 nm emission. The EC 50 and SEM were determined using GraphPad Prism 7.0.
Stage Specific Activity Assay (SSA): The SSA was performed as described previously [34]. Briefly, P. falciparum Dd2 cultures were tightly synchronized by magnetic isolation of schizonts using MACS (Miltenyi Biotec, Auburn, CA, USA) column [35] followed by 5% sorbitol (w/v) treatment [36]. Synchronized cultures were closely monitored by periodic Giemsa staining to identify the time of reinvasion. Six hours post invasion, cultures were plated into a 96 well plate, and compound was added to the 6 HPI treatment wells. At 12 h intervals, intraerythrocytic stage development was monitored via smears for Giemsa staining, and sample collection and fixing for flow cytometry. At each interval, a separate set of wells were likewise treated with compound for 18 and 30 HPI studies. For Giemsa staining of thin smears, a minimum of 1000 RBCs were counted, and the number of ring, trophozoite, and schizont infected RBCs was recorded. The most predominant stage for each smear was imaged using Leica DMi8 microscope and DMX-99 Digital camera.
Flow cytometry was performed on fixed, permeabilized, and YOYO-1 stained samples using CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA) as described previously [37]. Samples were gated to the RBC population and at least 100,000 events per well were recorded.